DNAzyme-aptamer electrochemical biosensor for Pb(II) and Hg(II) determination: robust validation in food matrices and...

A dual-recognition electrochemical biosensing platform was developed for the sensitive and selective determination of trace Pb(II) and Hg(II) in food and water matrices. The design integrates a Pb(II)-responsive DNAzyme and an Hg(II)-responsive aptamer on a graphene oxide-gold nanoparticle (GO-AuNP) modified gold electrode so that the two targets generate opposite impedance trends on the same interface. Structural analyses confirmed uniform AuNP decoration on GO sheets and stable probe immobilization through Au-S bonding. Under optimized conditions, Pb(II) promoted DNAzyme-mediated substrate cleavage and decreased the charge-transfer resistance, whereas Hg(II) induced T-Hg-T mediated aptamer folding and increased the impedance signal. The biosensor exhibited linear responses from 1.0 pM to 10 nM for Pb(II) and from 10 pM to 100 nM for Hg(II), with calculated limits of detection of 0.32 pM and 2.8 pM, respectively. The work clarifies that the analytical signal was obtained by measuring the same electrode before and after target incubation and that simultaneous exposure to Pb(II) and Hg(II) produced a net ΔR_ct dominated by partial signal compensation rather than frequency-resolved deconvolution. The platform also showed high selectivity against competing metal ions, same-electrode repeatability below 4%, between-batch fabrication precision below 5%, and storage stability above 92% over 28 days. Fit-for-purpose validation was performed in tap water, river water, seawater, milk, fish, and spinach samples, yielding recoveries of 91.2-108.0% and good agreement with ICP-MS. These results support the use of the present GO-AuNP/DNAzyme-aptamer architecture as a practical screening platform for trace heavy-metal monitoring in complex real samples.